1. Chapter 15 Biotechnology
(Sections ) 1

2. 15.1 Personal DNA Testing
Personal DNA testing companies identify a person’s unique array of single-nucleotide polymorphisms (SNPs) – some of which are related to risk of diseases such as Alzheimer’s
Personal genetic testing may soon revolutionize medicine by allowing physicians to customize treatments on the basis of an individual’s genetic makeup

3. Personal DNA Testing
This chip reveals which versions of 906,600 SNPs occur in the individual’s DNA
Figure 15.1 Personal genetic testing. Right, a SNP-chip. Personal DNA testing companies use chips like this one to analyze their customers’ chromosomes for SNPs. This chip, shown actual size, reveals which versions of 906,600 SNPs occur in the individual’s DNA.

4. ABC Video: DNA Mystery: Human Chimeras

5. ABC Video: Family Ties: Paternity Testing

6. 15.2 Cloning DNA
Researchers use restriction enzymes to cut up DNA, then bond the fragments together using DNA ligase
Fragments with complementary tails (“sticky ends”) stick together when their matching tails base-pair
restriction enzyme
Bacterial enzyme used to cut specific nucleotide sequences in DNA

7. Recombinant DNA
DNA fragments from different organisms combine to make a hybrid molecule: recombinant DNA
recombinant DNA
A DNA molecule that contains genetic material from more than one organism

8. Making Recombinant DNA (1)
A restriction enzyme recognizes a specific base sequence in DNA from any source
Figure 15.2 Using restriction enzymes to make recombinant DNA.

9. Making Recombinant DNA (2)
The enzyme cuts DNA from two sources into fragments
The enzyme leaves sticky ends
Figure 15.2 Using restriction enzymes to make recombinant DNA.

10. Making Recombinant DNA (3)
When DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other
Figure 15.2 Using restriction enzymes to make recombinant DNA.

11. Making Recombinant DNA (4)
DNA ligase joins the base-paired DNA fragments
Molecules of recombinant DNA result
Figure 15.2 Using restriction enzymes to make recombinant DNA.

12. restriction enzyme (cut)
Making Recombinant DNA
restriction enzyme (cut)
mix
DNA ligase
(paste)
A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source. 1
The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends. 2
When the DNA fragments from the
two sources are mixed together, matching sticky ends base-pair with each other. 3
DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result. 4
Figure 15.2 Using restriction enzymes to make recombinant DNA.
Fig. 15.2, p. 220

13. restriction enzyme (cut)
Making Recombinant DNA
A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source. 1
restriction enzyme (cut)
The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends. 2
mix
When the DNA fragments from the
two sources are mixed together, matching sticky ends base-pair with each other. 3
DNA ligase
(paste)
DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result. 4
Figure 15.2 Using restriction enzymes to make recombinant DNA.
Stepped Art
Fig. 15.2, p. 220

14. Animation: Restriction Enzymes

15. DNA Cloning DNA cloning mass produces specific DNA fragments
Fragments to be copied are inserted into plasmids or other cloning vectors and inserted into host cells such as bacteria
Host cells divide and make identical copies (clones) of the foreign DNA

16. Key Terms
DNA cloning
Set of procedures that uses living cells to make many identical copies of a DNA fragment
plasmid
Of many bacteria and archaeans, a small ring of nonchromosomal DNA replicated independently of the chromosome
cloning vector
A DNA molecule that can accept foreign DNA, be transferred to a host cell, and get replicated in it

17. Plasmid Cloning Vectors

18. Plasmid Cloning Vectors
Kpn l Sph l Pst l Bam Hl Eco RI Sal l Acc l Xho l Xba l Bst XI Sac l
Not l
pDrive Cloning Vector
3.85 kb
Figure 15.3 Plasmid cloning vectors. A Micrograph of a plasmid. B A commercial plasmid cloning vector. Restriction enzyme recognition sequences are indicated on the right by the name of the enzyme that cuts them. Researchers insert foreign DNA into the vector at these sites. Bacterial genes (gold) help researchers identify host cells that take up a vector with inserted DNA. This vector carries two antibiotic resistance genes and the lac operon (Section 10.5). A B
Fig. 15.3, p. 220

19. Plasmid Cloning Vectors
Figure 15.3 Plasmid cloning vectors. A Micrograph of a plasmid. B A commercial plasmid cloning vector. Restriction enzyme recognition sequences are indicated on the right by the name of the enzyme that cuts them. Researchers insert foreign DNA into the vector at these sites. Bacterial genes (gold) help researchers identify host cells that take up a vector with inserted DNA. This vector carries two antibiotic resistance genes and the lac operon (Section 10.5).
Fig. 15.3a, p. 220

20. Plasmid Cloning Vectors
Figure 15.3 Plasmid cloning vectors. A Micrograph of a plasmid. B A commercial plasmid cloning vector. Restriction enzyme recognition sequences are indicated on the right by the name of the enzyme that cuts them. Researchers insert foreign DNA into the vector at these sites. Bacterial genes (gold) help researchers identify host cells that take up a vector with inserted DNA. This vector carries two antibiotic resistance genes and the lac operon (Section 10.5).
Fig. 15.3b, p. 220

21. DNA Cloning

22. plasmid cloning vector
DNA Cloning
A A restriction enzyme cuts
a specific base sequence in chromosomal DNA and in
a plasmid cloning vector.
chromosomal
DNA fragments
chromosomal DNA
B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA.
recombinant plasmid
plasmid cloning vector
cut
plasmid
C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids.
Figure 15.4 An example of cloning. Here, a fragment of chromosomal
DNA is inserted into a plasmid.
Fig. 15.4, p. 221

23. plasmid cloning vector
DNA Cloning
plasmid cloning vector
chromosomal DNA
A A restriction enzyme cuts
a specific base sequence in chromosomal DNA and in
a plasmid cloning vector.
cut
plasmid
chromosomal
DNA fragments
B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA.
recombinant plasmid
C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids.
Figure 15.4 An example of cloning. Here, a fragment of chromosomal
DNA is inserted into a plasmid.
Stepped Art
Fig. 15.4, p. 221

24. ANIMATION: Formation of recombinant DNA
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25. cDNA Cloning
Researchers who study eukaryotic genes and their expression work with mRNA transcripts of genes
RNA can’t be cloned directly – reverse transcriptase, a viral enzyme, is used to transcribe single-stranded RNA into complementary DNA (cDNA) for cloning

26. Key Terms reverse transcriptase
A viral enzyme that uses mRNA as a template to make a strand of cDNA
cDNA
DNA synthesized from an RNA template by the enzyme reverse transcriptase

27. cDNA Cloning (1)
A strand of cDNA, is assembled on an mRNA template:

28. cDNA Cloning (2)
DNA polymerase removes RNA and copies the cDNA into a second strand of DNA, resulting in a double-stranded DNA copy of the original mRNA:

29. Key Concepts DNA Cloning
Researchers routinely make recombinant DNA by cutting and pasting together DNA from different species
Plasmids and other vectors can carry foreign DNA into host cells

30. ANIMATION: Base-pairing of DNA fragments
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31. ANIMATION: How to Make cDNA
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32. 15.3 From Haystacks to Needles
DNA libraries are sets of cells that host cloned DNA fragments
A genomic library collectively contains all DNA in a genome
A cDNA library contains only those genes being expressed at the time the mRNA was harvested
DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments

33. Key Terms
DNA library
Collection of cells that host different fragments of foreign DNA, often representing an organism’s entire genome
genome
An organism’s complete set of genetic material

34. Isolating Genes
Researchers use probes that match a targeted DNA sequence to identify cells with a specific DNA fragment
A probe mixed with DNA from a library base-pairs with (hybridizes to) the targeted gene
Base pairing between nucleic acids from different sources is called nucleic acid hybridization

35. Key Terms
probe
Short fragment of DNA labeled with a tracer such as a radioactive isotope
Designed to hybridize with a nucleotide sequence of interest
nucleic acid hybridization
Base-pairing between DNA or RNA from different sources

36. Nucleic Acid Hybridization

37. Nucleic Acid Hybridization
A Individual bacterial cells from a DNA library are spread over the surface of a solid growth medium. The cells divide repeatedly and form colonies—clusters of millions of genetically identical descendant cells.
B A piece of special paper pressed onto the surface of the growth medium will bind some cells from each colony.
C The paper is soaked in a solution that ruptures the cells and releases their DNA. The DNA clings to the paper in spots mirroring the distribution of colonies.
Figure 15.5 Nucleic acid hybridization. In this example, a radioactive probe helps identify a bacterial colony that contains a targeted sequence of DNA.
D A probe is added to the liquid bathing the paper. The probe hybridizes (base-pairs) with the spots of DNA that contain complementary base sequences.
E The bound probe makes a spot. Here, one radioactive spot darkens x-ray film. The position of the spot is compared to the positions of the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested.
Fig. 15.5, p. 222

38. ANIMATION: Use of a radioactive probe
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39. PCR
The polymerase chain reaction (PCR) uses primers and heat-resistant DNA polymerase to mass-produce a particular section of DNA without having to clone it in living cells
polymerase chain reaction (PCR)
Method that rapidly generates many copies of a specific section of DNA
primer
Short, single strand of DNA designed to hybridize with a DNA fragment

40. Multiplication by PCR
PCR can be used on any sample of DNA with at least one molecule of a target sequence
Essentially any sample containing DNA can be used, even one sperm, a hair left at a crime scene, or a mummy
Each cycle of a PCR reaction doubles the number of copies of a section of DNA – thirty cycles can make a billion copies

41. Two Rounds of PCR (1)
DNA template is mixed with primers, nucleotides, and heat- tolerant Taq DNA polymerase

42. Two Rounds of PCR (1) targeted section
DNA template (blue) is mixed with primers (pink), nucleotides, and heat-tolerant Taq DNA polymerase. 1
Figure 15.6 Two rounds of PCR. Each cycle of this reaction can double the number of copies of a targeted section of DNA. Thirty cycles can make a billion copies.
Fig , p. 223

43. Two Rounds of PCR (2)
When the mixture is heated, the double-stranded DNA template separates into single strands – when it is cooled, primers base-pair with template DNA

44. Two Rounds of PCR (2)
When the mixture is heated, the double-stranded DNA template separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.
Figure 15.6 Two rounds of PCR. Each cycle of this reaction can double the number of copies of a targeted section of DNA. Thirty cycles can make a billion copies. 2
Fig , p. 223

45. Two Rounds of PCR (3)
Taq polymerase begins DNA synthesis at primers, so complementary DNA strands form on single-stranded templates

46. Two Rounds of PCR (3)
Figure 15.6 Two rounds of PCR. Each cycle of this reaction can double the number of copies of a targeted section of DNA. Thirty cycles can make a billion copies.
Taq polymerase begins DNA synthesis at the primers, so complementary strands of DNA form on the single-stranded templates. 3
Fig , p. 223

47. Two Rounds of PCR (4)
The mixture is heated again; double-stranded DNA separates into single strands
When it is cooled, primers basepair with old and new DNA strands

48. Two Rounds of PCR (4)
Figure 15.6 Two rounds of PCR. Each cycle of this reaction can double the number of copies of a targeted section of DNA. Thirty cycles can make a billion copies.
The mixture is heated again, and the double-stranded DNA separates
into single strands. When it is cooled, some of the primers base-pair
with the template DNA. The copied DNA also serves as a template. 4
Fig , p. 223

49. Two Rounds of PCR (5)
Each round of PCR reactions doubles the number of copies of the targeted DNA section

50. Two Rounds of PCR (5)
Figure 15.6 Two rounds of PCR. Each cycle of this reaction can double the number of copies of a targeted section of DNA. Thirty cycles can make a billion copies.
Each round of PCR reactions can double the number of copies of the targeted DNA section. 5
Fig , p. 223

51. ANIMATION: Polymerase chain reaction (PCR)
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52. Key Concepts Finding Needles in Haystacks
DNA libraries, hybridization, and PCR are techniques that allow researchers to isolate and make many copies of a fragment of DNA they want to study

53. ANIMATION: Polymerase chain reaction
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54. 15.4 DNA Sequencing DNA sequencing reveals the order of bases in DNA
The entire genomes of several organisms have now been sequenced
DNA sequencing
Method of determining the order of nucleotides in DNA
DNA polymerase partially replicates a DNA template
Produces a mixture of DNA fragments of different lengths
Fragments are separated by electrophoresis

55. Electrophoresis
Electrophoresis separates fragments by length into bands
Electric field pulls DNA fragments through semisolid gel
Fragments of different sizes move at different rates
Shorter fragments move through the gel faster than longer fragments do
electrophoresis
Technique that separates DNA fragments by size

56. 5 Steps in DNA Sequencing
1. Sequencing depends on dideoxynucleotides to terminate DNA replication – each is labeled with a colored pigment

57. 5 Steps in DNA Sequencing
dideoxynucleotides
Figure 15.7 DNA sequencing, in which DNA polymerase is used to incompletely replicate a section of DNA. 1
Fig , p. 224

58. 5 Steps in DNA Sequencing
2. DNA polymerase uses a section of DNA as a template to synthesize new strands of DNA – synthesis of each new strand stops when a dideoxynucleotide with a tracer is added
3. At the end of the reaction, the mixture contains many incomplete copies of the original DNA

59. 5 Steps in DNA Sequencing
4. Electrophoresis separates DNA fragments into bands according to length – all DNA strands in each band end with the same dideoxynucleotide, and are the same color
5. A computer detects and records the color of successive bands on the gel – the order of colors represents the sequence of the template DNA

60. DNA Sequencing

61. DNA Sequencing DNA template 2 3 4
Figure 15.7 DNA sequencing, in which DNA polymerase is used to incompletely replicate a section of DNA. 4 5
Fig , p. 224

62. 3D Animation: Gene Sequencing

63. The Human Genome Project
The human genome consists of about 3 billion bases
Hoping to patent the sequence, Craig Venter’s company, Celera Genomics, invented faster methods of sequencing genomic DNA using supercomputers
By 2003, the human genome sequence was completed – but governments decided the sequence could not be patented

64. Human Genome Sequencing
Figure 15.8 Human genome sequencing. Left, some of the supercomputers used to sequence the human genome at Venter’s Celera Genomics in Maryland. Information in Celera’s SNP database is the basis of many new genetic tests. Right, a human DNA sequence, raw data.

65. Key Concepts DNA Sequencing
Sequencing reveals the linear order of nucleotides in DNA
Comparing genomes offers insights into human genes and evolution
An individual can be identified by unique parts of their DNA